Quantitative and qualitative analysis of pulmonary arterial hypertension fibrosis using wide-field second harmonic generation microscopy

We demonstrated that wide-field second harmonic generation (SHG) microscopy of lung tissue in combination with quantitative analysis of SHG images is a powerful tool for fast and label-free visualization of the fibrosis pathogenesis in pulmonary arterial hypertension (PAH). Statistical analysis of the SHG images revealed changes of the collagen content and morphology in the lung tissue during the monocrotaline-induced PAH progression in rats. First order statistics disclosed the dependence of the collagen overproduction on time, the second order statistics indicated tightening of collagen fiber network around blood vessels and their spreading into the alveolar region. Fourier analysis revealed that enhancement of the fiber orientation in the collagen network with PAH progression was followed with its subsequent reduction at the terminating phase of the disease. Proposed approach has potential for assessing pulmonary fibrosis in interstitial lung disease, after lung(s) transplantation, cancer, etc.


FFT method description
OI is calculated through the long-to-short ellipse axis ratio obtained via FFT analysis of the ROI [S1]. The transformed image from each ROI was binarized applying threshold, computed by the Otsu method [S2].
Despite the presence of some residual noise, a binary image with a significant elliptical structure was obtained. A predefined 2-D circular averaging filter for remaining noise removal (radius = 3 px) was applied. The received ellipse was approximated by second order curve using a least squares fitting method [S3].

Gray-Level Co-occurrence Matrix for SOS parameters evaluation
In this study, the image intensity is quantified by 256 levels. The 256×256 Gray-level Co-occurrence Matrix (GLCM) ( , , ) shows how often two pixels of i-th and j-th levels occur in the image at distance d. For a rectangular image having × pixels the GLCM is defined by the following equation [S4]: where ( , ) is the intensity of a pixel with coordinates ( , ).

Morphometric analysis and morpho-functional parameters, determined in animals with PAH
Morphometric analysis of the myocardium in animals with PAH showed the following changes [S5]. After 2 and 4 weeks, the wall thickness of the pancreas was statistically significantly higher than the control (p<0.05). After 1 month, the thickness of the pancreas progressively decreased and had no significant differences by the 8 th week of observation. At the same time, the transverse size of the cavity of the right ventricle (CRV) and the diameter of the trunk of the pulmonary artery (PA) significantly increased compared with the control group (p<0.05) (see Tables S4 and S5).    [3,2; 3,8] Notes: * -the presence of significant differences in values according to the Mann-Whitney test in comparison with the control group (p<0.05) RVC -transverse dimension of the cavity of the right ventricle PPP -transverse dimension of the cavity of the right atrium trunk LA -diameter of the trunk of the pulmonary artery Morphometric analysis of pulmonary vessels revealed the following changes [S6]. The development of MCT-induced PAH was accompanied by a significant increase in the thickness of the intima of small pulmonary capillaries, starting from the 2 nd week, and the thickness of the medial layer -by 8 weeks of the experiment (Tables S6). Notes: MTI -Media thickening index ITI -Intima thickening index We did not measure pulmonary artery pressure invasively. Hemodynamic parameters were assessed using an echocardiographic study (EchoCG) of the rat heart by indirect parameters, taking into account the technical difficulties in the determination of the hemodynamic parameters of the right heart due to the high heart rate of rats. The study was performed on an iE33 device (Philips Ultrasound, Bothell WA, USA) using a 12 MHz transducer according to generally accepted methods in M mode (one-dimensional echocardiography) and B mode (two-dimensional echocardiography).
EchoCG assessment of the studied parameters recorded a decrease (p<0.05) of the thickness of the interventricular septum, the posterior wall of the left ventricle in systole after 8 weeks of the experiment compared with the control group. Along with this, a decrease in the ejection fraction, shortening fraction, stroke volume of the left ventricle (p<0.05) was recorded at the same time of the experiment (Table S7).
The transverse size of the right ventricle gradually increased during the observation (p<0.05), and the thickness of the interventricular septum in systole increased after 2 weeks and did not differ from the control group (p<0.05) after 8 weeks.